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Open AccessResearch Metallic nickel nano- and fine particles induce JB6 cell apoptosis through a caspase-8/AIF mediated cytochrome c-independent pathway Address: 1 Pathology and Physio

Open Access

Research

Metallic nickel nano- and fine particles induce JB6 cell apoptosis

through a caspase-8/AIF mediated cytochrome c-independent

pathway

Address: 1 Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV, 26505, USA, 2 Graduate Center for Toxicology, College of Medicine, the University of Kentucky, Lexington, KY, 40515, USA and

3 Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV, 26505, USA

Email: Jinshun Zhao - fyq9@cdc.gov; Linda Bowman - llb2@cdc.gov; Xingdong Zhang - xaz5@cdc.gov; Xianglin Shi - xshi5@email.uky.edu;

Binghua Jiang - bhjiang@hsc.wvu.edu; Vincent Castranova - vic1@cdc.gov; Min Ding* - mid5@cdc.gov

* Corresponding author

Abstract

Background: Carcinogenicity of nickel compounds has been well documented However, the carcinogenic effect

of metallic nickel is still unclear The present study investigates metallic nickel nano- and fine particle-induced

apoptosis and the signal pathways involved in this process in JB6 cells The data obtained from this study will be

of benefit for elucidating the pathological and carcinogenic potential of metallic nickel particles

Results: Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we found that metallic

nickel nanoparticles exhibited higher cytotoxicity than fine particles Both metallic nickel nano- and fine particles

induced JB6 cell apoptosis Metallic nickel nanoparticles produced higher apoptotic induction than fine particles

Western-blot analysis showed an activation of proapoptotic factors including Fas (CD95), Fas-associated protein

with death domain (FADD), caspase-8, death receptor 3 (DR3) and BID in apoptotic cells induced by metallic

nickel particles Immunoprecipitation (IP) western blot analysis demonstrated the formation of the Fas-related

death-inducing signaling complex (DISC) in the apoptotic process Furthermore, lamin A and beta-actin were

cleaved Moreover, we found that apoptosis-inducing factor (AIF) was up-regulated and released from

mitochondria to cytoplasm Interestingly, although an up-regulation of cytochrome c was detected in the

mitochondria of metallic nickel particle-treated cells, no cytochrome c release from mitochondria to cytoplasm

was found In addition, activation of antiapoptotic factors including phospho-Akt (protein kinase B) and Bcl-2 was

detected Further studies demonstrated that metallic nickel particles caused no significant changes in the

mitochondrial membrane permeability after 24 h treatment

Conclusion: In this study, metallic nickel nanoparticles caused higher cytotoxicity and apoptotic induction than

fine particles in JB6 cells Apoptotic cell death induced by metallic nickel particles in JB6 cells is through a

caspase-8/AIF mediated cytochrome c-independent pathway Lamin A and beta-actin are involved in the process of

apoptosis Activation of Akt and Bcl-2 may play an important role in preventing cytochrome c release from

mitochondria to the cytoplasm and may also be important in the carcinogenicity of metallic nickel particles In

addition, the results may be useful as an important reference when comparing the toxicities of different nickel

compounds

Published: 20 April 2009

Journal of Nanobiotechnology 2009, 7:2 doi:10.1186/1477-3155-7-2

Received: 21 January 2009 Accepted: 20 April 2009 This article is available from: http://www.jnanobiotechnology.com/content/7/1/2

© 2009 Zhao et al; licensee BioMed Central Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nickel is a widely distributed metal that is industrially

applied in many forms The high consumption of various

nickel products inevitably leads to occupational and

envi-ronmental pollution [1] Carcinogenicity of nickel

com-pounds has been well documented [2-4] However, the

carcinogenic effect of metallic nickel is still unclear [5]

Evidence indicates that various nickel compounds, but

not metallic nickel, cause pulmonary inflammation,

fibrosis, emphysema, and cancer [6] The International

Agency for Research on Cancer (IARC), therefore,

classi-fied all nickel compounds as human carcinogens in 1990

[7] The available epidemiological studies on the

carcino-genicity of metallic nickel are limited by inadequate

expo-sure information, low expoexpo-sures, short follow-up periods,

and small numbers of cases [8] But evidences from

stud-ies in experimental animals suggest that metallic nickel is

reasonably anticipated to be a human carcinogen [5]

The metallic nickel nanoparticle is a product with many

new characteristics, which include a high level of surface

energy, high magnetism, low melting point, high surface

area, and low burning point Therefore, it can be widely

used in modern industries [9] However, these same

prop-erties of metallic nickel nanoparticles may present unique

potential health impact [10] Based on the fact that TiO2

nanoparticles are more toxic than TiO2 fine particles [11],

the pathological effects of nickel compounds and metallic

nickel may also depend on their particle size Nickel

com-pound (acetate)-induced apoptosis has been reported in

Chinese hamster ovary cells [12] and T cell hybridoma

cells [13] But the mechanism of cell death induced by

metallic nickel nano- and fine particles has not been

clearly elucidated

Apoptosis is a highly regulated process that is involved in

pathological conditions [14] Diseases may be caused by

a malfunction of apoptosis An inefficient elimination of

mutated cells may favor carcinogenesis [15] However,

excessive apoptosis was shown to contribute to

pulmo-nary fibrosis in mice [16] Furthermore, enhanced

apop-tosis may indirectly trigger compensatory cell

proliferation to ensure tissue homeostasis and promote

the fixation of mutagenic events Evidence indicates that

apoptosis is also involved in pulmonary disorders, such as

acute lung injury, diffuse alveolar damage, and idiopathic

pulmonary fibrosis [16,17] Therefore, the apoptotic

properties may be important in the mechanisms of

path-ogenicity and carcinpath-ogenicity induced by the metallic

nickel particles

Accordingly, the aim of the present study is to compare

the cytotoxicity and apoptosis induced by metallic nickel

nano- and fine particles, and to elucidate the mechanisms

of cell death induced by these particles in vitro.

Methods

Materials

Metallic nickel nanoparticles, average size 80 nm, were purchased from Inframat Advanced Materials, LLC (Farm-ington, CT) Metallic nickel fine particles, average size of

3 μm, were purchased from Sigma-Aldrich (Milwaukee, WI) Eagle's minimal essential medium (EMEM) was obtained from Lonza (Walkersville, MD) Fetal bovine serum (FBS), trypsin, pencillin/streptomycin and L-glutamine were purchased from Life Technologies, Inc (Gaithersburg, MD) YO-PRO-1 [YP, 1 mM solution in dimethyl sulfoxide (DMSO)] and propidium iodide (PI, 1.0 mg/ml in water) were purchased from Invitrogen (Carlsbad, CA) Anti-h/m caspase-8 antibody was obtained from R&D systems (Minneapolis, MN) Total Akt (Akt), phospho-Akt (p-Akt, ser 473), BID, and cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Beverley, MA) All other antibodies were obtained from Santa Cruz Biotechnology Co (Santa Cruz, CA) Cell proliferation kit I (MTT assay kit) was obtained from Roche Applied Science (Penzberg, Germany) Mito-chondria Staining Kit was purchased from Sigma-Aldrich (Saint Louis, MO)

Preparation of metallic nickel nano- and fine particles

Stock solutions of metallic nickel nano- or fine particles were prepared by sonification on ice using a Branson Son-ifier 450 (Branson Ultrasonics Corp., Danbury, CT) in sterile PBS (10 mg/ml) for 30 sec, then kept on ice for 15 sec and sonicated again for a total of 3 min at a power of

400 W Before use, these particles were diluted to a designed concentration in fresh culture medium All sam-ples were prepared under sterile conditions

Surface area and size distribution measurements

Surface area of metallic nickel particles was measured using the Gemini 2360 Surface Area Analyzer (Mircomer-itics; Norcross, GA) with a flowing gas technique accord-ing to the manufacturer's instructions The size distribution of metallic nickel particles was detected using scanning electron microscopy (SEM) Briefly, metallic nickel particles were prepared by sonification Then, the samples were diluted in double-distilled water and air dried onto a carbon planchet Images were collected on a scanning electron microscope (Hitachi S-4800; Japan) according to the manufacturer's instructions Optimas 6.5 image analysis software (Media Cybernetics; Bethesda, MD) was used to measure the diameter of metallic nickel particles

Cell culture

Mouse epidermal JB6 cells were maintained in 5% FBS EMEM containing 2 mM L-glutamine and 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin) at standard culture conditions (37°C, 80%

humidified air, and 5% CO2) For all treatments, cells

were grown to 80% confluence

Cytotoxicity assay

Cytotoxicity of metallic nickel nano- or fine particles to

JB6 cells was assessed by a MTT assay kit following the

manufacturer's instructions Briefly, cells were plated in

100 μl EMEM at a density of 104 cells/well in a 96 well

plate The cells were grown for 24 h and treated with

vari-ous concentrations of metallic nickel particles After 24 h

incubation, 10 μl MTT labeling reagent was added in each

well and the plates were further incubated for 4 h

After-ward, 100 μl solubilization solution was added to each

well and the plate was incubated overnight at 37°C The

optical density (OD) of the wells was measured at a

wave-length of 575 nm with reference of 690 nm using an ELISA

plate reader Results were calibrated with OD measured

without cells

Detection of apoptosis

YP staining was used to determine if cell death induced by

metallic nickel particles was apoptotic Briefly, JB6 cells

were seeded onto a 24-well plate overnight Then, cells

were treated with/without various concentrations of

metallic nickel nano- or fine particles for 24 h Before

microscopy, YP was added into the cultures (10 μg/ml) for

1 h Then, cells were washed two times with EMEM

medium Apoptotic cells were monitored using a

fluores-cence microscope (Axiovert 100 M; Zeiss, Germany)

Per-centage of cells exhibiting apoptosis was calculated

Identification of apoptosis

Dual staining using YP and PI was used to distinguish

between apoptosis and necrosis as described by Debby

and Boffa [18,19] with some modifications JB6 cells were

seeded onto a 24-well plate and incubated overnight

Then, cells were treated with/without various

concentra-tions of metallic nickel nano- or fine particles One hour

later, YP and PI were added into the cultures with a final

concentration of 10 μg/ml and 1 μM, respectively The

progression of cell death in the living cultures was

moni-tored at different time points on a fluorescence

micro-scope (Axiovert 100 M) YP stained cells were detected

with blue excitation filter PI stained cells were measured

by green excitation filter

Western blot analysis

Briefly, cells were plated onto a 6-well plate The cultures

were grown 24 h and then starved in 0.1% FBS EMEM

overnight Cells were treated with/without metallic nickel

nano- or fine particles After treatment, the cells were

extracted with 1× SDS sample buffer supplemented with

protease inhibitor cocktail (Sigma-Aldrich) Protein

con-centrations were determined using the bicinchoninic acid

method (Pierce; Rockford, IL) Equal amounts of proteins

were separated by 4–12% Tris glycine gels Immunoblots for expression of Fas, FADD, caspase-8, DR3, death recep-tor 6 (DR6), tumor necrosis facrecep-tor-receprecep-tor 2 (TNF-R2), caspase-3, caspase-6, caspase-9, BID, cleaved BID, Bcl-2,

BAX, cytochrome c, AIF, beta-actin, and lamin A were

detected Experiments were performed three or more times, and equal loading of protein was ensured by meas-uring total Akt, and alpha- or beta-tubulin expression

To prepare the subcellular fractionation, cells were washed twice with cold PBS Then, cells were lysed in 100

μl of cold isolation buffer A (20 mM Hepes/10 mM KCl/

supplemented with protease inhibitor cocktail and 250

mM sucrose After incubating on ice for 15 min, the cells were broken by passing through 22-gauge needles 25 times The lysate was centrifuged at 800 × g for 5 min to remove unbroken cells and nuclei The supernatant was then re-centrifuged (10,000 × g, 30 min, 4°C) to obtain a pellet The resultant supernatant was the cytosolic fraction and the pellet contained mitochondria The cytosolic frac-tion was diluted using 100 μl of 2× SDS sample buffer The mitochondrial pellet was resuspended in 1× SDS sam-ple buffer

IP western blot analysis

After treatment, JB6 cells were lysed in buffer B (20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, and 10 μl/ml protease inhibitor cocktail) for 15 min at 4°C Lysates were

centri-fuged at 25,000 × g for 15 min Protein concentrations of

the supernatants were determined Equal amounts of pro-teins were immunoprecipitated overnight with rabbit anti-caspase-8 antibody (1:200) at 4°C The supernatant was further incubated with 20 μl of protein A/G-agarose slurry for 3 h at 4°C Beads were pelleted, washed three times in buffer B, and finally boiled in 1× SDS sample buffer Proteins were separated by 4–12% Tris glycine gels Fas and FADD proteins were detected as described in west-ern blot analysis

Detection of mitochondrial membrane permeability

JB6 cells were seeded onto a 24-well plate overnight Cells were treated with/without metallic nickel nano- or fine particles for 24 h Changes of mitochondrial membrane permeability were evaluated using a mitochondrial stain-ing kit (JC1 stainstain-ing) accordstain-ing to the manufacturer's instructions Briefly, a staining mixture was prepared by mixing the staining solution with an equal volume of the EMEM medium Cells were incubated in the staining mix-ture (0.4 ml/well) for 30 min at 37°C in a humidified

washed two times in medium, followed by addition of fresh medium Mitochondrial membrane permeability

was monitored on a fluorescence microscope (Axiovert

100 M)

Statistical analysis

Data are presented as means ± standard errors (S.E.) of n

experiments/samples Significant differences were

deter-mined using R software or the Student's t-test Significance

was set at p ≤ 0.05.

Results

Surface area and size distribution of metallic nickel

particles

To measure the surface area and size distribution of nickel

particles, Gemini 2360 Surface Area Analyzer and

scan-ning electron microscopy were used, respectively The

average surface area of metallic nickel nanoparticles was

4.36 m2/g compared to 0.40 m2/g for fine particles The

average size distribution of metallic nickel nano- and fine

particles is 92.32 nm and 3.34 μm, respectively (Table 1)

SEM images of the metallic nickel particles

Metallic nickel nano- or fine particles were prepared by

sonification Then, the samples were diluted in

double-distilled water and air dried onto a carbon planchet SEM

images were captured on a scanning electron microscope

(Figure 1A and 1B)

Effects of metallic nickel particles on cell viability and

apoptotic induction

To determine whether there is a difference in the

cytotox-icity induced by different sizes of metallic nickel particles,

various concentrations (0.1–20 μg/cm2) of metallic nickel

nano- or fine particles were used to study the effects on

cell viability in JB6 cells by MTT assay Treatment of JB6

cells with metallic nickel particles for 24 h caused a

dose-dependent cytotoxicity (Figure 2A) Cytotoxicity induced

by metallic nickel nanoparticles was significantly higher

than that induced by fine particles

To study the apoptosis induced by metallic nickel

nano-or fine particles, YP staining was used JB6 cells were

treated with various concentrations of metallic nickel

nano- or fine particles from 0.1 to 20 μg/cm2 for 24 h

Results indicated that both metallic nickel nano- and fine

particles induced JB6 cell apoptosis (Figure 2B) The

per-centages of apoptotic cells were significantly higher in cells treated with nanoparticles than fine particles

increase in apoptosis induced by nanoparticles compared

to fine particles

Identification of apoptosis induced by metallic nickel particles

To distinguish between apoptosis and necrosis induced by metallic nickel nano- or fine particles, a dual staining assay using YP and PI was applied The results showed that both metallic nickel nano- and fine particles (data not shown) could induce JB6 cell apoptosis demonstrated by the positive staining of YP at an early exposure time (24 h)

cm2, 48 h) resulted in necrosis or late apoptosis demon-strated by the positive staining of both YP and PI (Figure 3A and 3B)

Effects of metallic nickel particles on caspase-8, Fas, FADD, DR3, DR6, TNF-R2, p-Akt, DISC, lamin A, beta-actin, BID, Bcl-2, and BAX

Previous studies have demonstrated that apoptosis acti-vates an upstream protease caspase-8 [20,21] In this study, JB6 cells were treated with 20 μg/cm2 of metallic nickel nano- or fine particles for 30, 60, 120, and 180 min Protein expressions were detected by western-blot Results indicated that caspase-8 was activated by these particles (Figure 4A)

Two important signals are known to be involved in apop-tosis, which include the TNF and the Fas-Fas ligand-medi-ated pathways Both involve the TNF receptor family coupled to extrinsic signals [22] To investigate the involvement of extrinsic signals in the apoptotic process induced by metallic nickel particles, expression of the TNF family members of Fas, FADD, DR3, DR6, and TNF-R2 was examined Results demonstrated that metallic nickel particles activated Fas, FADD and DR3 However, no obvi-ous change was found in the protein expression of DR6 or TNF-R2 (Figure 4A)

Akt is a well-characterized member of PI3 kinase-medi-ated signaling pathways, regulating cell growth, apopto-sis, as well as other cellular responses Akt activation inhibits apoptosis by phosphorylating the Bcl-2 related proteins In addition, Akt activation is sufficient to inhibit

the release of cytochrome c from mitochondria and the

alterations in the inner mitochondrial membrane poten-tial [23] In this study, results indicated that both metallic nickel nano- and fine particles induced Akt phosphoryla-tion in a time-dependent manner (Figure 4A)

Table 1: Surface area and size distribution of metallic nickel

particles

Nickel fine particles Nickel nanoparticles

Surface area (m 2 /g) 0.4 ± 0.01 4.36 ± 0.02

Average size 3.34 ± 0.67 (μm) 92.32 ± 29.69 (nm)

Surface area was determined by gas absorption and particle size by

scanning electron microscopy Values are means ± S.E of six

independent assays.

As caspase-8 activation was detected, we further

deter-mined the involvement of the DISC formation in the

process of apoptosis induced by metallic nickel particles

The interaction between Fas and FasL results in the

forma-tion of the DISC, which consist of Fas, FADD, and

cas-pase-8 [22] To investigate the formation of DISC, IP

western blot was used JB6 cells were treated with 20 μg/

cm2 metallic nickel nano- or fine particles for 30, 60, 120,

and 180 min Anti-caspase-8 IP revealed an interaction of

Fas and FADD with caspase-8, demonstrating DISC

for-mation and the initiation of Fas-induced apoptotic

path-way (Figure 4B)

The cellular morphology associated with the apoptotic

process has been well characterized by membrane

bleb-bing, formation of apoptotic bodies, and chromosome

condensation These apoptotic changes are the result of

the cleavage of cellular proteins, such as lamin and actin

[24,25] In this study, JB6 cells were treated with 20 μg/

cm2 metallic nickel nano- or fine particles for 1, 3, 6, and

8 h Western blot revealed that the cleavages of lamin A

and beta-actin were detected as early as 1 h post-exposure

Both particles induced lamin A cleavages in a

time-dependent manner (Figure 4C)

BID, a proapoptotic member of the Bcl-2 family, is a phys-iological substrate of caspase-8 which causes mitochon-drial damage [26] The results demonstrated that metallic nickel nano- or fine particles induced BID cleavage in a time-dependent manner Interestingly, Bcl-2, an anti-apoptotic protein, was up-regulated BAX, a proanti-apoptotic member of Bcl-2 family, was down-regulated (Figure 4D)

Effects of metallic nickel particles on AIF, cytochrome c, caspase-3, -6, and -9

AIF is a recently characterized proapoptotic mitochon-drial protein [27] It is normally confined to the mito-chondrial inter membrane space After release from mitochondria into the cytoplasm, AIF can stimulate cell apoptosis [28] To test the effects of metallic nickel parti-cles, JB6 cells were treated with 20 μg/cm2 nano- or fine particles for 1, 3, 6, and 8 h Western blots revealed that both nano- and fine particles induced mitochondrial AIF up-regulation and release from mitochondria to the cyto-plasm after 1 h treatment (Figure 5A)

Cytochrome c is an important apoptotic factor in the

intrinsic apoptotic pathway which is released into the cytoplasm from the mitochondria in response to

proap-SEM images of metallic nickel particles

Figure 1

SEM images of metallic nickel particles SEM images of metallic nickel fine (A) or nanoparticles (B) were captured on a

scanning electron microscope

Effects of metallic nickel particles on cell viability and apoptotic induction

Figure 2

Effects of metallic nickel particles on cell viability and apoptotic induction JB6 cells were exposed to various

con-centrations of metallic nickel nano- or fine particles for 24 h Cell viability was detected by MTT assay Significantly less viability

was observed in cells treated with nanoparticles compared to fine particles analyzed by R software (p < 0.05) Data shown are

means ± S.E of four independent assays (A) Apoptosis induced by metallic nickel nano- or fine particles was detected by YP staining (B, 10× magnification) Metallic nickel nanoparticles induced more apoptosis than fine particles at 0.5 and 5 μg/cm2

ana-lyzed by Student's t-test (p < 0.05) indicated by * (C) Data shown are means ± S.E of three independent assays.

Identification of apoptosis induced by metallic nickel nanoparticles

Figure 3

Identification of apoptosis induced by metallic nickel nanoparticles JB6 cells were seeded onto 24-well plate and

incubated overnight Cells were treated with/without metallic nickel nanoparticles Continuous monitoring of apoptosis and

necrosis was conducted by using a dual fluorescence dye assay after 24 h treatment (A) or 48 h treatment (B).

optotic stimuli [29] To investigate the possible

involve-ment of cytochrome c release in the process of apoptosis

induced by metallic nickel particles, JB6 cells were treated

with 20 μg/cm2 of metallic nickel nano- or fine particles

for 1, 3, 6, 8 h Western blot analysis indicated that

cyto-chrome c was not released from the mitochondria into the

cytoplasm although metallic nickel particles could induce

cytochrome c up-regulation (Figure 5B).

Caspases are a family of cysteine proteases which play

essential roles in apoptosis, necrosis and inflammation

[30] Eleven caspases have so far been identified in

humans There are two types of apoptotic caspases:

initia-tor caspases and effecinitia-tor caspases Initiainitia-tor caspases (e.g

caspase-8) cleave inactive pro-forms of effector caspases,

thereby activating them Effector caspases (e.g caspase-3

and -6) in turn cleave other protein substrates resulting in the apoptotic process Since activation of caspase-8 was detected, we next examined the possible involvement of caspase-3, -6, and -9 in the process of apoptosis induced

by metallic nickel particles Results indicated that metallic nickel particles induced only a slight activation of

caspase-3, -6, and -9 Interestingly, caspase-3 precursor was signif-icantly up-regulated by metallic nickel particles (Figure 5C)

Effects of metallic nickel particles on mitochondrial membrane permeability

Mitochondrial membrane permeability change is a hall-mark for apoptosis [31] JB6 cells were treated with/with-out various concentrations of metallic nickel particles for

24 h Mitochondrial membrane permeability was

evalu-Effects of metallic nickel particles on caspase-8, Fas, FADD, DR3, DR6, TNF-R2, p-Akt, DISC, lamin A, beta-actin, BID, Bcl-2, and BAX

Figure 4

Effects of metallic nickel particles on caspase-8, Fas, FADD, DR3, DR6, TNF-R2, p-Akt, DISC, lamin A, beta-actin, BID, Bcl-2, and BAX Cells were treated with 20 μg/cm2 metallic nickel particles for 30, 60, 120, and 180 min

Expressions of caspase-8, Fas, FADD, DR3, DR6, TNF-R2, and p-Akt were analyzed by western blot (A) To investigate the formation of DISC, IP western blot was used (B) Cells were treated with metallic nickel particles for 1, 3, 6, and 8 h Effects of metallic nickel particles on lamin A, beta-actin, and Bcl-2 family were detected by western blot (C and D).

Effects of metallic nickel particles on AIF, cytochrome c, and caspase-3, -6, and -9

Figure 5

Effects of metallic nickel particles on AIF, cytochrome c, and caspase-3, -6, and -9 To determine the effects of

metallic nickel particles on AIF, cytochrome c, and caspase-3, -6, and -9, JB6 cells were seeded onto a 6-well plate After 24 h

incubation, cells were starved in 0.1% FBS EMEM overnight Then, cells were treated with 20 μg/cm2 metallic nickel particles

for 1, 3, 6, and 8 h Western blot analysis was used to detect the effects of metallic nickel particles on AIF (A), cytochrome c

(B), and caspase-3, -6, and -9 (C).

ated using a mitochondrial staining kit according to the

manufacturer's instructions The results indicated that

nei-ther metallic nickel nano- nor fine particles induced any

significant change in the mitochondrial membrane

per-meability compared to negative control after 24 h

treat-ment Positive control cells treated with 0.5 μl

valinomycin/well for 1 h showed a significant effect on

the mitochondrial membrane permeability (Figure 6A

and 6B)

Discussion

Nickel and nickel compounds are widely used in

indus-tries In occupational settings, workers are exposed to a

variety of nickel compounds, nickel alloys, as well as

metallic nickel About 10% of all the primary nickel

pro-duced is used in metallic form [5] Human exposure to

nickel or its compounds has the potential to produce a

variety of pathological effects The most important adverse health effects due to nickel exposure are skin aller-gies, lung fibrosis, and lung cancer [7]

With the increase use of nanoparticles in modern indus-tries, inhaled nanoparticles are increasingly being recog-nized as a potential health threat [32] It is well known that the toxicity of particles to the lung in both occupa-tional and environmental settings is not only related to exposure but also to the particle size Accordingly, metal-lic nickel nanoparticles may be more toxic than the con-ventional metallic nickel fine particles

In the present study, results show that both metallic nickel nano- and fine particles induce a dose-related increase in cytotoxicity in JB6 cells after 24 h exposure In addition, metallic nickel nanoparticles are more toxic than fine

par-ticles Our in vitro finding is in agreement with the previ-ous in vivo reports that metallic nickel nanoparticles are

more toxic on the bronchoalveolar lavage fluid in rats than metallic nickel fine particles [9] Apoptosis is a pro-grammed form of cell death which is now widely recog-nized as being of critical importance in health and disease Although studies have demonstrated that nickel com-pounds induce cell apoptosis [12], the molecular path-ways have not been well investigated It is generally accepted that cell death can either result in apoptosis or necrosis Our results suggest that both metallic nickel nano- and fine particles induce JB6 cell death through apoptosis, but not necrosis, at early exposure time in a cer-tain dose range With the treatment duration prolonged or treatment dose enhanced, both metallic nickel nano- and fine particles can induce JB6 cells necrosis or late apopto-sis For the quantification of apoptosis, we carried out YP staining to determine the apoptotic cells induced by vari-ous concentrations of metallic nickel particles The results showed that both nano- and fine particles induce JB6 cell apoptosis in a dose response manner after 24 h treatments

in a dose range of 0.1–20 μg/cm2 At concentrations of 5

nano-particles was 4 fold higher than fine nano-particles Our results suggest that both metallic nickel nano- and fine particles are cytotoxic in JB6 cells, while metallic nickel nanoparti-cles show higher cytotoxicity and apoptosis induction than fine particles In an inhalation study in rats,

Ober-dörster et al found TiO2 nanoparticles to be more

inflam-matory than fine particles [11] When normalized to surface area, the authors found that the dose-response curves for the nano- and fine particles were similar, sug-gesting that the pulmonary inflammation was mediated

by surface effects In the present study, surface area of metallic nickel nanoparticles is 11-fold greater than fine particles However, metallic nickel nanoparticles exhib-ited potency for toxicity and apoptosis which was some-what less than 11-fold greater than fine particles

Effect of metallic nickel particles on mitochondrial membrane

permeability

Figure 6

Effect of metallic nickel particles on mitochondrial

membrane permeability JB6 cells were treated with

var-ious concentrations of metallic nickel nano- or fine particles

for 24 h A mitochondrial staining kit was used to detect the

mitochondrial membrane permeability induced by metallic

nickel fine (A) or nanoparticles (B).